A Novel Post-translational Modification in Nerve Terminals: O-Linked

ARTICLE pubs.acs.org/jpr

A Novel Post-translational Modification in Nerve Terminals: O-Linked N-Acetylglucosamine Phosphorylation Mark E. Graham,*,† Morten Thaysen-Andersen,‡ Nicolai Bache,† George E. Craft,† Martin R. Larsen,§ Nicolle H. Packer,‡ and Phillip J. Robinson† †

Cell Signalling Unit, Children’s Medical Research Institute, The University of Sydney, Westmead, Australia Protein Research Group, Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark ‡ Department of Chemistry and Biomolecular Sciences, Biomolecular Frontiers Research Centre, Macquarie University, Sydney, Australia §

bS Supporting Information ABSTRACT: Protein phosphorylation and glycosylation are the most common post-translational modifications observed in biology, frequently on the same protein. Assembly protein AP180 is a synapse-specific phosphoprotein and O-linked beta-N-acetylglucosamine (O-GlcNAc) modified glycoprotein. AP180 is involved in the assembly of clathrin coated vesicles in synaptic vesicle endocytosis. Unlike other types of O-glycosylation, O-GlcNAc is nucleocytoplasmic and reversible. It was thought to be a terminal modification, that is, the O-GlcNAc was not found to be additionally modified in any way. We now show that AP180 purified from rat brain contains a phosphorylated O-GlcNAc (O-GlcNAc-P) within a highly conserved sequence. O-GlcNAc or O-GlcNAc-P, but not phosphorylation alone, was found at Thr-310. Analysis of synthetic GlcNAc-6-P produced identical fragmentation products to GlcNAc-P from AP180. Direct O-linkage of GlcNAc-P to a Thr residue was confirmed by electron transfer dissociation MS. A second AP180 tryptic peptide was also glycosyl phosphorylated, but the site of modification was not assigned. Sequence similarities suggest there may be a common motif within AP180 involving glycosyl phosphorylation and dual flanking phosphorylation sites within 4 amino acid residues. This novel type of protein glycosyl phosphorylation adds a new signaling mechanism to the regulation of neurotransmission and more complexity to the study of O-GlcNAc modification. KEYWORDS: AP180, glycosyl phosphorylation, N-acetylglucosamine phosphorylation, synaptic vesicle endocytosis, phosphorylation, graphite, mass spectrometry, nerve terminals

’ INTRODUCTION The attachment of β-N-acetylglucosamine (GlcNAc) via Ser and Thr hydroxyl groups is common on nuclear and cytoplasmic proteins with diverse functions.1,2 The intracellular location of O-linked GlcNAc (O-GlcNAc) is commensurate with its role as a reversible glycosylation that is involved in dynamic signaling events,3,4 similar to protein phosphorylation. O-GlcNAc is added to proteins by O-GlcNAc-transferase (OGT)5 and removed by β-N-acetylglucosaminidase/O-GlcNAcase.6 This is in contrast to protein phospho-regulation, where hundreds of protein kinases and protein phosphatases are responsible for the dynamic modification. O-GlcNAc modified proteins are commonly found to be phosphoproteins. Some well studied O-GlcNAc modified proteins have a complex interplay with phosphorylation.1,7
9 Thus, O-GlcNAc modification and phosphorylation may compete for the same site (Ser or Thr), O-GlcNAc modification and phosphorylation can occupy adjacent sites, or one type of modification can regulate the occupancy of the other modification at a distant site. r 2011 American Chemical Society

OGT mRNA is highly abundant in brain and OGT expression is enriched in presynaptic nerve terminals, as are O-GlcNAc modified proteins.10 It is not surprising that many of the hundreds of known O-GlcNAc modified proteins are found in nerve terminals. However, it is not clear how O-GlcNAc regulates the function of these proteins. O-GlcNAc mainly occurs on cytoskeletal related proteins such as tau,11 neurofilament L and M,12 CMRP-2 and MAPs,13 suggesting a role in neuron morphology via cytoskeletal regulation. Also, OGT has been detected surrounding synaptic vesicles.10 The occurrence of O-GlcNAc on synapsins,7 piccolo, and bassoon14 suggests a role in synaptic vesicle exocytosis, via vesicle tethering and cytoskeleton organization. Modification of endophilin-A2, phosphatidylinositol-binding clathrin assembly protein (CALM)15 and clathrin assembly protein AP18016 by O-GlcNAc suggests a role in synaptic vesicle endocytosis (SVE). SVE involves the retrieval of synaptic vesicles from the plasma membrane. This process proceeds by different modes, some of Received: November 5, 2010 Published: April 18, 2011 2725

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Figure 1. AP180 is O-GlcNAc-P modified, in its disordered clathrin and AP-2 binding domain, with dual flanking phosphorylation sites. (A) AP180 consists of an AP180 N-terminal homology domain (ANTH) and a disordered C-terminal domain with multiple clathrin and AP-2 binding motifs. Thr310 was found to be O-GlcNAc/O-GlcNAc-P modified in rat AP180. Ser-306, 313, 621, and 627 were phosphorylated. (B) Sequence containing Thr310-O-GlcNAc-P and the dual flanking phosphorylation sites conserved in most available vertebrate sequences of AP180. Within AP180 there exists a similar sequence that is likely to be O-GlcNAc-P modified. The UniprotKB or NCBI accession number is provided (XL is Xenopus laevis, XT is Xenopus tropicalis).

which are mediated by the formation of a clathrin coat.17 AP180 (UniProtKB/Swiss-Prot Q05140) is one of the adaptor proteins that bind lipid and clathrin to assist in the initial formation of the clathrin coat. At the later stages of clathrin-mediated endocytosis, AP180 becomes a coat constituent and its presence has an influence on the size and shape of the nascent synaptic vesicles.18
20 AP180 is one of a number of SVE proteins that are regulated by stimulus-dependent dephosphorylation in nerve terminals.21 Following nerve terminal depolarization, the resultant calcium influx stimulates the protein phosphatase calcineurin to dephosphorylate these proteins. The removal of specific inhibitory phosphosites has been shown to promote protein
protein interactions that stimulate SVE.22,23 AP180 has two domains: an AP180 N-terminal homology (ANTH) lipid-binding domain and a C-terminal clathrin assembly domain that has direct interactions with clathrin and the adapter protein complex AP-2 (Figure 1A). There are multiple copies of short sequence motifs that bind either AP-2 or clathrin. AP180 may bind to AP-2 through DΦF motifs (where Φ is a hydrophobic amino acid residue) or through FxDxF motifs (where x is any amino acid residue).24 Multiple DLL motifs, which are perhaps a degenerate form of the original clathrin box motif

(L(L/I)(D/E/N)(L/F)(D/E)),25 are thought to be responsible for mediating clathrin binding.26 Many of these short motifs also conform to a revised clathrin box motif (pLΦpΦp, where p is a polar amino acid residue).27 Recently, two of these degenerate motifs in AP180 were shown to directly bind the clathrin terminal domain.28 Phosphorylation sites have been found on AP180 from mouse brain at Tyr-15,29 at Ser-313 in two studies30,31 and at Thr-312, Ser-316 and Ser-600 in synaptosomes (isolated nerve terminals).31 A recent study32 detected all of these phosphosites in mouse brain as well as phosphorylation at Ser-70, Ser-107, Ser-296, Ser300, Ser-303, Thr-321, Thr-324, Ser-325, Ser-335, Ser-593 and Ser-594. Phospho-Ser-594 was also detected on rat AP180.33 Some of these mouse and rat phosphosites are expected to exist in other organisms because of the high sequence homology between known vertebrate AP180 genes. However, apart from phospho-Ser-594, none of these phosphosites have been independently validated. Phospho-Ser-107 could have originated from the identical sequence in the ANTH domain of the ubiquitous CALM, which is highly homologous to the ANTH domain of brain specific AP180. Both O-GlcNAc and phosphorylation have been detected on the AP180 clathrin assembly domain.16 The stoichiometry of 2726

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Journal of Proteome Research O-GlcNAc modification was reported as one O-GlcNAc per protein molecule,16 but the exact site of the O-GlcNAc modification was not determined. We now confirm AP180 is O-GlcNAc modified at Thr-310. Free GlcNAc can be phosphorylated by GlcNAc kinase34 to produce GlcNAc-6-phosphate (GlcNAc-6-P). GlcNAc-6-P was found as a component of complex carbohydrates in bovine colostrums.35 However, there have been no reports that Ser/Thr-O-GlcNAc can be further modified in any way, such as by phosphorylation. We have now identified Thr310 as a site that can be occupied by either O-GlcNAc or O-GlcNAc-phosphate (O-GlcNAc-P) and identified a second potential site with similar modification, probably Thr-625. Here, we present the characterization of this novel PTM, O-GlcNAc-P, on purified AP180 from rat brain nerve terminals using mass spectrometry.

’ MATERIALS AND METHODS Materials

N-Acetyl-D-glucosamine-6-phosphate disodium salt and N-acetyl-D-glucosamine-1-phosphate disodium salt were from Sigma (Sydney, Australia). Antarctic alkaline phosphatase was from Genesearch (Gold Coast, Australia). Milli-Q water was used in all experiments (Milli-Q UF PLUS; Millipore, Sydney, Australia). Titanium dioxide beads were obtained from a disassembled Titansphere column (GL Sciences, Tokyo, Japan). Trypsin (porcine, modified) was from Promega (Sydney, Australia). The plasmid expressing GST-alpha adaptin appendage domain (mouse, amino acids 701
938) was as previously reported.22 Purification of AP180 from Synaptosomes

Crude (P2) synaptosomes36 were prepared from rat brains as described previously.22 Briefly, synaptosomes in low calcium Krebs-like buffer (118.5 mM NaCl, 4.7 mM KCl, 1.18 mM MgCl2, 0.1 mM K2HPO4, 20 mM HEPES, 10 mM glucose, with 0.1 mM calcium, pH 7.4) were incubated for 30 min at 37 °C. The synaptosomes were lysed in 25 mM Tris, pH 7.4, containing 1% Triton X-100, 150 mM NaCl, 1 mM EGTA, 2 mM EDTA, 50 mM NaF, 20 μg/mL leupeptin, 1 mM PMSF and EDTA-free protease inhibitor cocktail (Roche, Sydney, Australia). AP180 was purified from the lysates using its affinity to GST-alpha adaptin appendage domain bound to glutathione sepharose beads as described previously.22 The beads were washed extensively, eluted in 2 concentrated SDS sample buffer and resolved on SDS gels. The extracted AP180 was run in a 1 mm thick large format gel (20 cm, length) using the BioRad Protean II system. A 7.5
15% acrylamide and 0.2
0.4% bis-acrylamide linear gradient SDS-PAGE gel was used. Note that rat AP180 isoform 1 contains a 19 amino acid insert found only in rat. The numbering of the AP180 amino acid residues used throughout this work is from the more conserved isoform 2 sequence. Tryptic Digestion and Phosphopeptide Enrichment

AP180 gel bands were excised from the gels, diced to 1 mm3 and destained by vortexing with three 1 mL aliquots of 50% acetonitrile in water until the bands were translucent (approximately 4 h) and digested with trypsin in 50 mM triethylammonium bicarbonate at 37 °C for 16 h. The solution was then made up to 50% acetonitrile and the tryptic peptides were extracted after 15 min of vortexing. A second extraction was obtained with 80% acetonitrile solution with a further 15 min of vortexing. Samples were dried in vacuo (ALPHA-RVC IR, CHRIST, Germany). The AP180 phosphopeptides and O-GlcNAc-P

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modified peptides were enriched from the mixture using a titanium dioxide microcolumn as described previously.37 Nanoliquid Chromatography Mass Spectrometry

The titanium dioxide-enriched, AP180 phosphopeptides and O-GlcNAc-P modified peptides were analyzed by LC
MS/MS using information dependent acquisition (QSTAR XL quadrupole-TOF MS, AB SCIEX, Foster City, CA) and reversed phase chromatography (LC Packings Ultimate HPLC system, Dionex, Netherlands) with a 50 μm inside diameter C18 column at 100 nL/min, as previously described.37 The sample was rerun using fixed precursor ion selection to allow more time to accumulate signal for particular spectra. The samples were also analyzed on an 1100 Agilent nanoflow HPLC system connected to an MSD Trap XCT Ultra II (Agilent Technology, Santa Clara, CA) electron transfer dissociation (ETD) MS. The sample was loaded using the same C18 chromatography columns and conditions except that the loading on the precolumn was at 3 μL/min for 10 min and the gradient was from 100% solvent A (water with 0.1% (v/v) formic acid) to 40% solvent B (water with 80% (v/v) acetonitrile and 0.1% (v/v) formic acid) over 58 min. The automated gain control was set to 500 000 and the maximum accumulation time was set to 150 ms. Additional settings were: SmartFrag on (Ampl 30
200%), fragmentation time 40 ms, ETD reaction time 85 ms, reactant accumulation time 50 ms and reactant temperature 60 °C. To separate the glycopeptides and improve sequence information the samples were also analyzed using graphite HPLC with ETD MS. Separation was performed using a Hypercarb porous graphitised carbon column (5 μm particle size, 320 μm  10 cm, Thermo Scientific) equipped with a 0.5 μm peek filter (Upchurch, Oak Harbor,WA) on an Ultimate 3000 LC (Dionex) and analyzed using ESI
MS in positive polarity mode on an HCT 3-D ion trap (Bruker Daltonics) coupled directly to the LC. The graphite column was equilibrated in 100% solvent A (aqueous 0.1% (v/v) formic acid), the sample loaded and a gradient up to 60% (1%/min slope) solvent B (0.1% (v/v) formic acid in acetonitrile) was applied before washing the column in 90% B for 10 min. The column was heated to 55 °C during the run and a constant flow-rate of 5 μL/min was used. As above, fixed precursor ion scans were used over the entire HPLC run. An estimated 1 pmol of the O-GlcNAc/O-GlcNAc-P modified peptide AP180 305
320 was injected and m/z 610.3 was targeted as a fixed precursor ion. The m/z value was isolated using ( m/z 2 allowed window, ICC target 200 000 and maximum accumulation time of 50 ms. The ETD fragmentation was done with ICC reactant target 600 000, maximum accumulation time 200 ms and a reaction time of 150 ms. The fragments were scanned from m/z 150
2200. The modified peptides were further targeted by creating extracted ion chromatograms from the data using two fragment ions, m/z 400.2 (z4) and m/z 487.3 (z5), common to all the targets. The chromatograms were smoothed using the Gauss smoothing algorithm (2 s smoothing width, 1 cycle). The collision induced dissociation (CID) or ETD MS/MS spectra, for fixed precursor ions, were summed across the portion of the peak which was within 75% of the height of the peak apex, to produce high quality data for identification of the peptide (Analyst QS 1.1, AB SCIEX; DataAnalysis 3.2.121.0, Agilent or Compass DataAnalysis 4.0, Bruker Daltonik). The individual or summed CID spectra were subject to a threshold, centroided and then converted to peak lists using the script Mascot.dll version 2727

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16b25 for Analyst QS, in preparation for protein database searching. For CID data, the threshold for each spectrum was arbitrarily determined to exclude noise/background signals. For ETD data, the spectra were subject to a S/N threshold of 1, relative intensity threshold of 0.05% of base peak and absolute intensity threshold of 50, were centroided, copied to a text file as a peak list and converted to Mascot generic format. The pseudo MS3 spectra were obtained by infusing desalted38 AP180 tryptic digests or synthetic carbohydrates into the QSTAR XL with increased declustering potential (DP2 from 10 up to 20). The collision energy was held at 16 V for 10% of the acquisition time and at 30 V for 90% of the acquisition time to emphasize the lower m/z fragment ions. Protein Database Searching

The parameters for creating peak lists for multiple spectra in the mascot.dll version 16b25 script were: precursor mass tolerance for grouping was 1 unit of m/z, maximum number of cycles between groups was 10, minimum number of cycles per group was 1, all MS/MS data was centroided and deisotoped, peaks were removed if less than 0.1% of maximum and spectra were rejected if they had less than 8 peaks, attempt to determine charge state from survey scan was on and default precursor charge states were 2þ, 3þ, 4þ and 5þ. Database searching was performed using a local copy of Mascot version 2.2 (Matrix Science, London, U.K.). The searched database was the rat international protein index version 3.74 (39 708 sequences; 21 112 183 residues). The searches were done with some or all of the variable modifications; deamidation (NQ), phosphorylation (ST), HexNAc (T) and a custom modification: HexNAcPhospho (T). The precursor ion mass tolerance was 150 ppm and fragment ion tolerance was 0.1 Da for quadrupole-TOF MS data and 0.6 Da for ETD MS data. Enzyme specificity for tryptic digests was selected to trypsin with no missed cleavages. Mascot Scores or Expect (probability) values were not used as criteria for identification, although such data is provided in Supporting Information, Supplementary Table S1. Ultimately, all spectra were manually validated. This required assessing how well each spectrum matched the theoretical fragment ions of the peptide and if the signal intensity of each ion was appropriate and in some cases comparing nonmodified to modified peptide spectra. Novel or comparative spectra are supplied in Figures or in Supporting Information.

’ RESULTS AND DISCUSSION Detection and Verification of the O-GlcNAc-P Modification

Purified AP180 from rat brain synaptosomes was digested with trypsin. AP180 phosphopeptides were enriched from the digest using titanium dioxide chromatography to determine sites of phosphorylation on AP180. Our attention was drawn to a peptide with unusual properties. A triply charged peptide at m/z 610.3 was specifically enriched in the titanium dioxide eluate, indicating it was a phosphopeptide, but gave poor MS/MS information (Figure 2). Rather than showing the fragmentation characteristics of a phosphopeptide, the spectrum was typical of O-linked glycopeptides: (i) the precursor fragmented to produce a spectrum dominated by a low m/z ion (e.g., m/z 204, indicative of fragmented O-linked N-acetylhexosamine) and (ii) the remaining fragment ions described a nonmodified peptide. Yet, we confirmed that the peptide was also modified by phosphorylation since treatment with alkaline phosphatase resulted in a loss of 80 Da from the precursor molecular mass and the fragment ion

Figure 2. CID MS/MS spectrum of AP180 305
320 with additional mass of 283 Da. A titanium dioxide enriched peptide with a triply charged signal at m/z 610.3 was fragmented. The spectrum provided some fragment ions of unmodified AP180 305
320, but was dominated by low mass ions including a HexNAc reporter ion, m/z 204, and a strong signal 80 Da greater at m/z 284.

signal at m/z 284 (Supplementary Figure S1, Supporting Information). The ion at m/z 204 indicated that the glycan was O-linked N-acetylhexosamine. However, the more abundant signal at m/z 284 fully accounted for the difference between the nonmodified (M, 1545 Da) and modified AP180 305
320 (M, 1828 Da) peptide. The neutral mass difference of 283 Da did not match any known glycan except the GlcNAc-1-phosphoryl modification in the Unimod database (PhosphoHexNAc, accession #428). This phosphoryl glycosylation, found only on a slime mold protein,39 involves a phosphate sequestered in a phosphodiester bond between GlcNAc and Ser. A phosphate in this position would be sterically protected from removal by a phosphatase, which was not the case for AP180 (Supplementary Figure S1, Supporting Information). Therefore, the þ283 Da modification on AP180 was not GlcNAc-1-phosphoryl. To obtain more information on the identity of the signal at m/z 284, we obtained a pseudo MS3 fragmentation spectrum of this ion (Figure 3A). An AP180 tryptic digest was analyzed by nanoelectrospray ionization. The declustering potential was increased to intensify the production of the m/z 284 ion and it was then selected for fragmentation. The resulting spectrum had fragment ions indicative of neutral loss of water (
18 Da), acetic acid (
60 Da) and phosphoric acid (
98 Da) originating from hydroxyl, acetyl and phosphoryl functional groups, respectively. To confirm our hypothesis that the signal at m/z 284 was a molecular ion similar to phosphorylated GlcNAc, we compared the fragmentation of the sample of rat brain origin to synthetic GlcNAc-6-P (CAS Registry ID 102029
88
9, 301.2 Da). The MS/MS spectrum of synthetic GlcNAc-6-P is shown in Figure 3B. The spectra were nearly identical, apart from small differences in fragment ion intensity and some different signals near the level of the background noise. The similarity of the spectra allowed us to conclude that they are chemically and structurally indistinguishable by mass spectrometry, but the question remains as to the location of the phosphate group. GlcNAc-3-P and GlcNAc-4-P were not available for comparison to definitively determine the location of the phosphate on the in vivo sample. GlcNAc-1-P was the only other similar molecule available. The MS/MS spectrum of GlcNAc-1-P is shown in Figure 3C. In contrast to GlcNAc-6-P and the ion at m/z 284, the immediate neutral loss of phosphate from GlcNAc-1-P is favored over the loss of water. This further 2728

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Figure 3. Comparison of the fragmentation of the m/z 284 signal, from rat brain synaptosomes, with fragmentation of synthetic GlcNAc-6-P and GlcNAc-1-P. (A) MS/MS spectrum of m/z 284 from the in-source fragmentation of AP180 O-GlcNAc-P modified peptides in a pseudo MS3 experiment. (B) MS/MS spectrum of commercial GlcNAc-6-P. C. MS/MS spectrum of commercial GlcNAc-1-P. Both structural isomers have a monoisotopic molecular mass of 301.06. Postulated neutral losses (Da) and the formulas commonly associated with these neutral losses are shown.

demonstrates that the phosphate is not present in a diester linkage to AP180 since the fragment ions do not support this scenario. We conclude that the signal at m/z 284 is diagnostic for the presence of O-GlcNAc-P which has a phosphate at the 3, 4, or 6 position. There is no biological relevance to assert that any position of phosphate substitution on O-GlcNAc is more likely, except for the precedents that free GlcNAc can be phosphorylated at the 6 position34 and GlcNAc-6-P was found as a component of complex carbohydrates in bovine colostrums.35 We sought to determine the site of O-GlcNAc and O-GlcNAcP modification on AP180 305
320. No such information was obtained from the spectrum in Figure 2, which is typical of MS/MS spectra of peptides with labile O-linked glycans. Various methods have been employed to determine O-GlcNAc sites including enzymatic derivatization15 or β-elimination combined with chemical derivatization40 with MS detection. However, these methods were not suitable for preserving the heterogeneity of the AP180 peptide. That is, detecting the existence of O-GlcNAc or O-GlcNAc-P on the same peptide. The reliance on these methods could explain why O-GlcNAc-P has not been previously discovered, despite at least three previous targeted brain tissue analyses.14,15,40 The presence of both m/z 284 and 204 in

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Figure 4. Graphite HPLC separation and ETD MS/MS spectra of structural isomers of AP180 305
320 þ 283 Da reveals a new posttranslational modification to Thr-310. (A) The precursor ion with a triply charged signal at m/z 610.3 was isolated for the entire LC
MS/MS run. An extracted ion chromatogram created by targeting two fragment ions m/z 400.2 (z4) and m/z 487.3 (z5) common to all the peptides is shown. Chromatographic peaks are labeled P1
4. (B) The fragmentation spectrum created from P1 matched AP180 305
320 with O-GlcNAc modification at Thr-310 and phosphorylation at Ser-313. The difference between the fragment ions at c6 and c5 are equal to the molecular mass of a Thr residue modified with O-GlcNAc (304 Da) and the phosphorylated Ser-313 is located between the nonphosphorylated c8 and phosphorylated c10 ions (c9 is absent but not required for phosphosite localization). (C) The spectrum from the abundant P3, in A, matched AP180 305
320 with O-GlcNAc-P modification at Thr-310. The difference between the fragment ions at c6 and c5 are equal to the molecular mass of a Thr residue modified with O-GlcNAc-P (384 Da). P2 and P4, in A, were deamidated Asn-315 versions of P1 and P3 (precursor m/z 610.8), respectively (Supplementary Figure S2A and B, Supporting Information).

Figure 2 established that both O-GlcNAc and O-GlcNAc-P existed on the same AP180 sequence. Note that the data in Figure 3A verified that m/z 204 could not have arisen from fragmentation of m/z 284. We used ETD MS to clarify which forms of the modified peptide exist, by preserving the O-glycosyl linkages. To produce clear, single peptide ETD spectra, we first needed to separate the mixture of isobaric glycopeptides we were detecting after C18 HPLC (Figure 2). By changing to graphite 2729

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Journal of Proteome Research chromatography, we were able to separate the structural isomers. An LC-MS/MS experiment targeting m/z 610.3 showed four chromatographic peaks that had shared fragment ions from AP180 305
320 (Figure 4A). We were able to identify the peptide in each peak using ETD MS. The first eluting peak (P1, Figure 4A) was AP180 305
320 with Thr-310-O-GlcNAc and phospho-Ser-313 (Figure 4B). The second, low abundance eluting peak (P2) was the deamidated (Asn-315 to Asp/isoAsp) form of this same peptide (Supplementary Figure S2A, Supporting Information). The ETD spectrum of P3 (Figure 4C) had a difference of 384 Da between c5 and c6 ions showing conclusively for the first time, combined with other evidence from above, that threonine can be modified by O-GlcNAc-P. P4 was the deamidated version of this peptide (Supplementary Figure S2B, Supporting Information). Interestingly, when a proteolytic fragment of AP180, beginning at Ser-305 was previously sequenced by Edman degradation, Thr-310 could not be identified (“SSPATXVT”).41 Molecular mass alone cannot be used to identify the Thr-O-linked N-acetylhexosamine, since GlcNac and GalNAc are isobaric. However, previous work16 established that O-GlcNAc, and not O-GalNAc is present on AP180. We conclude that AP180 can be modified by O-GlcNAc and, at a higher abundance, by O-GlcNAc-P at Thr-310. The Unimod database (www.unimod.org) accession #428 was created for GlcNAc-1-phosphoryl but has been updated to include the O-GlcNAc-P modification, with specificity for Thr modification. These modifications are structural isomers. Thus they have an identical molecular formula and mass (C8H14NO8P, monoisotopic mass 283.045704 Da). GlcNAc-1-phosphoryl modification has been referred to as phosphoglycosylation,39 which is potentially confusing. The terms phosphoglycosylation and glycophosphorylation are ambiguous. We suggest that protein phosphoryl glycosylation should refer to the addition of glycans via a phospho-diester bond to the protein, such as GlcNAc-1-phosphoryl. Conversely, protein glycosyl phosphorylation should refer to the addition of O- or N-linked glycans that contain phosphorylated carbohydrates, such as in O-GlcNAc-P and mannosyl-6-P.42 The Sequence of O-GlcNAc-P Modification Flanked by Phosphorylation Is Potentially Repeated in AP180

We found no evidence that Thr-310 could also be directly phosphorylated, in a sharing arrangement, as has been demonstrated for O-GlcNAc sites on other proteins.8 Instead, we found that phosphorylation of flanking serine residues occurred. The phosphorylation at Ser-313 can be detected on AP180 in the presence (Figure 4B) or absence (Supplementary Figure S3, Supporting Information) of glycosylation at Thr-310. Using ETD MS we found that the AP180 305
320 peptide could be modified with O-GlcNAc-P at Thr-310 and also phosphorylated at Ser-313 (Supplementary Figure S4A, Supporting Information). Furthermore, the same peptide could be additionally phosphorylated at Ser-306 (Supplementary Figure S4B, Supporting Information). Derivatization of the N-terminal Ser-305 to allow detection of the b1 fragment ion was necessary to confirm that Ser-306, rather than the adjacent Ser-305, was phosphorylated (Supplementary Figure S5, Supporting Information). In conclusion, up to three phosphate groups, including the one protruding from O-GlcNAc, could be detected within the sequence of AP180 306-313. In our phosphopeptide-enriched sample, we detected another peptide that also produced diagnostic fragment ions at both m/z

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Figure 5. CID MS/MS spectrum of AP180 598
630 with additional mass of 283 Da from O-glycosylation compared to nonmodified AP180 598
630. (A) The titanium dioxide enriched peptide with a triply charged signal at m/z 1132.5 was fragmented. The spectrum provided some fragment ions of unmodified AP180 598
630 but was dominated by low mass ions including the HexNAc reporter ion at m/z 204 and a signal 80 Da greater at m/z 284 indicative of O-GlcNAc-P modification. (B) The spectrum in A is similar to the nonmodified MS/MS spectrum of AP180 598
630 (from the triply charged signal at m/z 1038.2), indicating they contain the same sequence, except that the peptide in A is O-glycosylated and phosphorylated.

204 and 284 (O-GlcNAc and O-GlcNAc-P fragmentation products). This large peptide was from a triply charged signal at m/z 1132.5 (3394.6 Da, Figure 5A). As above (Figure 2), the sequence information was poor. However, the available fragment ions indicated that the peptide was AP180 598
630 with either O-GlcNAc-P or O-GlcNAc and phospho-Ser-600. The spectrum had many of the same fragment ions in the MS/MS spectrum of nonphosphorylated AP180 598
630, shown in Figure 5B, and phospho-(Ser-600/627)-AP180 598
630, shown in Supplementary Figure S6A and B, Supporting Information. The phosphorylation sites near the C-terminal end of this peptide (the last 10 residues of AP180 598
630) were interesting because of the high sequence similarity to the O-GlcNAc-P modified AP180 305
320 (Figure 1B). The shorter phosphorylated semitryptic peptide AP180 616
630 was available for fragmentation in the phosphopeptide enriched sample. Chromatographic separation of two monophosphorylated AP180 616
630 peptides enabled confirmation of the phosphorylation at Ser-627 (Supplementary Figure S7A, Supporting Information) and the discovery of another phosphosite at Ser-621 (Supplementary Figure S7B, Supporting Information). We were not able to confirm the position of the O-GlcNAc/O-GlcNAc-P on AP180 598
630 due to poor fragment signals. However, the sequence 2730

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Journal of Proteome Research similarity suggests that Thr-625 would align well with the O-GlcNAc/O-GlcNAc-P site at Thr-310 and Ser-621 and Ser627 would align well with the proline-directed phosphosites (Pro at þ1 position) at Ser-306 and Ser-313 (Figure 1B). We conclude that there is the potential for a repeated motif of O-GlcNAc-P modification with dual flanking phosphosites. Phospho-Ser-306, Ser-621 and Ser-627 have not been found previously in any organism. Ten amino acid residues separates Ser-627 from a known clathrin terminal domain binding site,28 suggesting a possible role for AP180 glycosyl phosphorylation and flanking phosphorylation in mediating interaction with clathrin. The interaction or cooperation of O-GlcNAc-P with nearby phosphorylation sites offers a number of possibilities for the function of such a motif. Multiple phosphosites increase net negative charge and hydrophilicity. O-GlcNAc also increases hydrophilicity and therefore solubility. These combined effects may enable AP180 to reduce binding to proteins or other electronegative surfaces such as the membrane which would imply that these modifications could inhibit SVE. We used iTRAQ labeling to test whether various phosphorylated and O-GlcNAc/ O-GlcNAc-P modified versions of AP180 305
320 were responsive to a potassium depolarization stimulus in synaptosomes (data not shown). The small change that we measured was less than the error in the measurement. Therefore, these modifications are either not dynamic or only a very small pool of O-GlcNAcP modified AP180 is stimulated or we have not monitored an appropriate biological stimulus. As an alternative to inhibiting protein
protein interactions, the function of O-GlcNAc-P and flanking phosphorylation might be to allow binding to proteins which are attracted to phosphorylation or glycosyl phosphorylation. As examples, 14-3-3 proteins bind phosphoproteins43 and mannose-6-P is bound by the mannose-6-P receptor proteins.42 O-GlcNAc-P may also simply be a way for AP180 to achieve a particular conformation during its role in clathrin coat formation.

’ CONCLUSION We have discovered O-GlcNAc-P, a new type of protein glycosyl phosphorylation. This finding is significant given the potential for some of the hundreds of known O-GlcNAc modified sites to also be O-GlcNAc-P sites. O-GlcNAc-P might have been overlooked in many otherwise careful primary structure studies, as occurred in the original AP180 study.16 In the alternative scenario, O-GlcNAc-P could be unique to brain specific AP180 and crucial to forming the clathrin coat on synaptic vesicles. In the latter scenario, targeting this modification could potentially be an exquisitely specific way to therapeutically gain control of SVE, or some particular types of SVE. Questions arise as to how O-GlcNAc-P occurs. Our data suggests stepwise modification from O-GlcNAc to O-GlcNAc-P. However, we cannot rule out a one-step process. There is currently no evidence for the existence of UDP-GlcNAc-phosphate which would potentially be used in a similar way that UDPGlcNAc is used by OGT to add O-GlcNAc to proteins. Either scenario suggests that there may be a new glycosyltranferase/ kinase or new function for a known glycosyltransferase/kinase to be discovered. It would be surprising if the phosphate was not removable by an as yet undiscovered phosphatase. Targeting these undiscovered components may be a route to control of SVE or other potential O-GlcNAc-P modified proteins. It is also

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possible that O-GlcNAc is chemically phosphorylated by a reactive molecule as in protein pyrophosphorylation.44 Diagnostic ions exist that can be used to find other occurrences of O-GlcNAc-P, allowing further investigation of this modification and its function. However, the occurrence of O-GlcNAc-P near flanking phosphorylation sites, in AP180, is a barrier to quantitative study since it is difficult to monitor one particular modification among multiple modifications on the same tryptic peptide. Tools to monitor this novel modification are required to determine how it influences AP180 with a view to determining its specialized role in neurotransmission.

’ ASSOCIATED CONTENT

bS

Supporting Information Supplementary Table S1, list of identified peptides statistical information. Supplementary Figure S1, annotated spectra of phosphatase treated AP180 peptides. Supplementary Figure S2, annotated spectrum of deamidated, glycosylated and phosphorylated AP180 305
320. Supplementary Figure S3, annotated spectrum of monophosphorylated AP180 305
320. Supplementary Figure S4, annotated spectra of glycosylated and multiple phosphorylated AP180 305
320. Supplementary Figure S5, annotated spectrum confirming phosphorylation at Ser-306. Supplementary Figure S6, annotated spectrum confirming phosphorylation at Ser-600 and Ser-627. Supplementary Figure S7, annotated spectrum confirming phosphorylation at Ser-621. Peaks lists for each peptide mass spectrum are supplied in Mascot generic format. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*Dr. Mark E. Graham, Cell Signalling Unit, Children’s Medical Research Institute, Locked Bag 23, Wentworthville, NSW 2145, Australia, Tel. þ61-2-9687-2800; Fax. þ61-2-9687-2120; E-mail: [email protected].

’ ACKNOWLEDGMENT This work was supported by an the Mizutani Foundation for Glycoscience, an Australian Research Council Discovery Project, a Project Grant from the National Health and Medical Research Council of Australia (P.J.R. and M.E.G.) and a Career Development Award from the National Health and Medical Research Council of Australia (M.E.G). M.T.-A. was supported by The Danish Council for Independent Research | Natural Sciences. N.B. was supported by the Danish Agency for Science, Technology and Innovation. Additional support was from The Ian Potter Foundation and the Cancer Institute New South Wales. We thank our colleagues who have generously provided materials for this study. ’ REFERENCES (1) Hart, G. W.; Housley, M. P.; Slawson, C. Cycling of O-linked beta-N-acetylglucosamine on nucleocytoplasmic proteins. Nature 2007, 446 (7139), 1017–1022. (2) Wells, L.; Vosseller, K.; Hart, G. W. Glycosylation of nucleocytoplasmic proteins: signal transduction and O-GlcNAc. Science 2001, 291 (5512), 2376–2378. 2731

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